PROJECT SUMMARY/ABSTRACT Cardiovascular disease is the leading cause of death in the US. Research on the use of gene therapy for cardiovascular purposes is quickly advancing but face critical problems, such as off-target effects and host immune response against the vectors. Adeno-associated virus (AAV) is a promising gene delivery vector and has been used in more than 200 clinical trials worldwide. The goal of this project is to create an improved AAV- based vector with targeted delivery and to characterize the host immune response to this vector in comparison to unmodified AAV capsid. Our lab has created a protease-activatable AAV vector, called the provector, that can deliver transgenes to sites with elevated extracellular proteases. The provector is locked (?OFF? state) until cleaved by matrix metalloproteinases (?ON? state), which are elevated in diseases such as post-myocardial infarction cardiac remodeling, congestive heart failure, and atherosclerosis. The prototype provector, however, has incomplete de-targeting, which may lead to undesired side-effects. This project seeks to optimize provector de-targeting in the OFF state while maintaining transduction in the ON state. Additionally, the in vivo behavior of the optimized provector will be explored and key components in the immune response to this provector and the unmodified capsid will be identified. To achieve these objectives, two specific aims will be completed. First, the provector will be optimized for increased on-target activation and decreased off-target activity. To do so, a mosaic provector will be created in which some subunits will contain a strong peptide lock optimized for decreased transduction in the OFF state, while others will be wild-type to increase ON state activity. The virus?s physical properties and ON and OFF transduction profiles will be characterized in vitro. Second, key components in the immune response against AAV will be identified. Both the optimized provector and the wild-type AAV capsid will be administered to immune competent and several immune deficient strains of mice. The overall virus behavior and immune response generated will be measured after one administration and after re-administration. The responses of different mice strains will be compared to identify essential immune components and their effect on virus behavior and immune response. This research is significant because it will create a more effective protease- activatable provector and will offer a comparative study on the effect of various immune system components on anti-AAV immune response and virus behavior. As a result, this study will identify key components that can be targeted for the developing of immune-evasive vectors in the future. These results have significant implications on the development of targeted and immune-evasive vectors for clinical use, which will greatly advance the field of AAV-based gene therapy and create new treatments for a variety of diseases.